Beyond a PPR-RNA recognition code: Many aspects matter for the multi-targeting properties of RNA editing factor PPR56

Abstract

The mitochondrial C-to-U RNA editing factor PPR56 of the moss Physcomitrium patens is an RNA-binding pentatricopeptide repeat protein equipped with a terminal DYW-type cytidine deaminase domain. Transferred into Escherichia coli, PPR56 works faithfully on its two native RNA editing targets, nad3eU230SL and nad4eU272SL, and also converts cytidines into uridines at over 100 off-targets in the bacterial transcriptome. Accordingly, PPR56 is attractive for detailed mechanistic studies in the heterologous bacterial setup, allowing for scoring differential RNA editing activities of many target and protein variants in reasonable time. Here, we report (i) on the effects of numerous individual and combined PPR56 protein and target modifications, (ii) on the spectrum of off-target C-to-U editing in the bacterial background transcriptome for PPR56 and two variants engineered for target re-direction and (iii) on combinations of targets in tandem or separately at the 5'- and 3'-ends of large mRNAs. The latter experimentation finds enhancement of RNA editing at weak targets in many cases, including cox3eU290SF as a new candidate mitogenome target. We conclude that C-to-U RNA editing can be much enhanced by transcript features also outside the region ultimately targeted by PPRs of a plant editing factor, possibly facilitated by its enrichment or scanning along transcripts.

Fig 1. PPR56 and site-directed mutations in its DYW cytidine deaminase domain.

A. Makeup of PPR56 and its two native targets. PPR56 is a typical plant organelle RNA editing factor featuring a PLS-type PPR array with alternating P-, L- and S-type PPRs followed by extension motifs E1 and E2 and a terminal DYW cytidine deaminase domain. Typically, the most C-terminal PLS triplet of plant editing factors has a deviating consensus and is labeled P2-L2-S2. As suggested previously [88], to account for generally more loosely conserved N-terminal repeats, PPRs are numbered backwards with the terminal PPR S2-1 juxtaposed with position -4 upstream of the editing target cytidine converted into uridine. Shading of matches in green follows the PPR-RNA recognition code based on amino acid identities in positions 5 and L in P- and S-type PPRs: T/S+N:A, T/S+D:G, N+D:U, N+S:C, N+N:Y. The corresponding amino acid identities in the TPR-like E1 and E2 motifs are indicated in italics. PPR56 has two native editing targets in the mitochondria of Physcomitrium patens: nad4eU272SL and nad3eU230SL. Near-complete editing (>99%) is generally observed for the nad4eU272SL target, but lower editing (>70%) is variably observed for nad3eU230SL in planta, possibly as a result of different strains or cultivation conditions [38,39]. B. Mutations in the DYW domain of PPR56. Twelve conserved amino acid positions (see S1 Fig) in the DYW domain of PPR56 were selected for mutations and tested on both native targets nad4eU272SL and nad3eU230SL in the E. coli RNA editing assay system. RNA editing efficiencies are given as the mean of at least three biological replicates (independent primary E. coli clones) when RNA editing activity was detected. Initially identified absence of RNA editing for a construct was confirmed with at least one additional independent bacterial clone. All primary data for RNA editing assays are given in S1 Data. C. Mutations in the E1 and E2 motifs of PPR56. Positions 34 (‘last’) of the E1 and E2 motifs potentially juxtaposed with nucleotide positions -3 and -2 upstream of the edited cytidine have been mutated and tested on the two native targets of PPR56 with RNA editing remaining unaffected by the PPR56|E1:N34D mutant, but dropping dramatically for the PPR56|E2:K34A mutant.

Fig 2. PPR56 target mutations opposite of P- and S-type PPRs.

Mutations have been introduced upstream of the two native PPR56 editing targets nad4eU272SL and nad3eU230SL in positions juxtaposed with P- and S-type PPRs assumed to follow the PPR-RNA code rules for amino acid positions 5 and L. Ten target mutants investigated earlier [36] are indicated with asterisks at the respective percentages (e.g. for nad4eU272SL|a-16u, top left). Designation of PPRs, numbering of positions and shading in target sequences is as in Fig 1A. Average RNA editing activities from three replicates are given below individually mutated positions or next to multiple mutations (boxed). Primary data are listed in S1 Data.

Fig 3. Compensating and non-compensating PPR and target mutants.

Key positions ‘5’ or ‘Last’ have been altered in individual PPRs of PPR56 (red font) in attempts of re-targeting to modified target sequences with conceptually improved matches in individual positions (green shading) of native targets nad4eU272SL and nad3eU230SL, respectively. RNA editing activities are indicated for the individual PPR mutants next to the respective target position identities. A. Target mutations not rescued by corresponding PPR mutations. No re-gain of RNA editing activity is observed for PPR mutations S-13NS>ND, P-12NN>NS, S-7TD>TN, P2-3ND>NS, L2-2VD>ND and S2-1ND>NS (red cylinders) juxtaposed with nucleotide positions -16, -15, -10, -6, -5 and -4 upstream of the edited cytidine in either target. B. Target mutations at least partially rescued by corresponding PPR mutations. Moderate re-gains of RNA editing activity are observed for at least one of the two targets for PPR mutations S-10TD>TN, P-9TN>TD, P-6ND>TD, S-4TN>TD and S2-1ND>TD (blue cylinders) opposite of nucleotide positions -13, -12, -9, -7 and -4, respectively. The green cylinder and shading indicates the mutated PPR P2-3ND>NN with a conceptually relaxed selectivity for U over C in position -6. C. Double target mutant. A double mutant PPR56|S-7TD>TN|S-4TN>TD shows no activity on the native targets but can be rescued to different amounts by the corresponding g-10a|a-7g target double mutants. D. N-terminal PPR truncations of PPR56. Progressive truncation of the two or three terminal PPRs of PPR56 lead to moderate or more drastic reduction of RNA editing efficiencies, respectively.

Fig 4. Mutation of target positions opposite of L-type PPRs.

Target positions -14, -11 and -8 opposite of L-type PPRs L-11MD, L-8VD and L-5LD have been changed to the nucleotides present in the respective other native target of PPR56. Additional mutations to purines were introduced in positions -11 and -5 opposite of PPRs L-8VD and L2-2VD, which carry the same combination of amino acids in positions 5 and L and are mainly juxtaposed with cytidines in the targets. The strongest effects are seen for nad3eU230SL|a-11g and nad3eU230SL|c-5g abolishing RNA editing completely in the modified nad3 targets. Vice versa, a much stronger effect is seen for the triple mutant nad4eU272SL|a-14g|c-11a|u-8c in the nad4 target vs. the inverse changes nad3eU230SL|g-14a|a-11c|c-8u in the nad3 target.

Fig 5. Mutations around the RNA editing sites.

The two native targets of PPR56, nad4eU272SL and nad3eU230SL, feature identical nucleotides in positions -2 to +2 around the edited cytidines (uuCau). With the exception of the exchange u-1g eradicating RNA editing completely at both targets, other exchanges in the upstream region show different outcomes with nad4eU272SL|u-2g (31%) vs. nad3eU230SL|u-2g (0%) or the inverse pyrimidine exchanges in position -3 with no effect for nad4 editing but reduction to 22% for nad3. Changes in positions +1 and +2 do not affect editing of the nad4 target but reduce editing of nad3. The overall tolerance of the nad4 target region against mutations in positions -1, +1 and +2 allows to engineer all three artificial stop codon identities (red) or an artificial start codon (green) to be created by C-to-U RNA editing.

Fig 6. The influence of sequences further upstream of targets.

PPR56 editing targets were cloned with 17 bp of additional native sequence upstream of the region supposed to be ultimately targeted by the PPR array, with the C-terminal PPR S2-1 juxtaposed with position -4 upstream of the editing site. Progressive 5’-truncations of this upstream sequence to only eight, seven, five or one nucleotide matching the native target behind the SwaI cloning site (AUUUAAAU) place them in closer proximity to the upstream vector sequences (blue) with nucleotides not matching the native upstream sequences underlined. The shortening results in serially decreased RNA editing activity to 53% for the nad4 target. A yet stronger effect is seen for the nad3 target where a 5’-truncation retaining four native upstream nucleotides reduces editing to 18%. Replacing the AU-rich sequence upstream of positions -20 with a GC-rich sequence (red font) abolishes editing at the nad3eU230SL site altogether.

Fig 7. Off-target analyses.

Off-targets of PPR56, PPR56|S4TN>TD and PPR56|S10TD>TN in the E.coli transcriptome summarized with Weblogo [89]. Consensus profiles were created from the sequences of 119, 382 and 15 C-to-U RNA editing off-targets, weighted with their respective editing efficiencies. Additional off-targets requiring nucleotide shifts for better binding matches (14, 67 and 1, respectively) were excluded for clarity (S2 Data). Modified positions in the PPRs are displayed in red. The mutated PPRs have a clear preference to the nucleotides fitting best to the modified binding amino acid pair in positions 5 and L according to the PPR-RNA code. Nucleotide preferences in positions −3, −2 and −1 are highlighted in blue. Nucleotide preferences within the PPR stretch and opposite to P- or S- motifs are highlighted in green.

Fig 8. Combining different PPR56 targets.

To test for mutual influences of combined targets on the same transcript, a series of tandem constructs and a triplicate arrangement of nad3 targets was cloned in the multiple cloning site behind the PPR56 coding region. Shading highlights native targets nad3eU230SL (yellow) and nad4eU272SL (pink) and two off-targets identified in the E. coli background transcriptome in the transcripts of yegH (blue) and folD (grey). Numbers in parentheses indicate RNA editing efficiency observed in the off-target analysis and when cloned individually without the upstream nad4 target, respectively. The series of constructs with the nad4 upstream of the nad3 target includes the one with the GC-rich sequence upstream of the latter (red font) that had abolished nad3eU230SL editing altogether.

Fig 9. RNA editing target placement at the 5’- or 3’-end of a long mRNA.

A. The two native targets of PPR56 were placed separately into the previously used 3’-MCS downstream of the protein coding sequence (blue lines) and into the newly created 5’-MCS (red lines) in pET41Kmod2 (S2 Fig) in both alternative combinations. Editing efficiencies for single targets inserted in 5‘ MCS are given in brackets. Cloning is done via NotI-PacI in the 5’-MCS and via SwaI-AscI in the 3’-MCS. B. The tandem combination of the two targets previously tested in the 3’-MCS was now also tested in the 5’-MCS.

Fig 10. Off-targets in different cloning positions.

Five off-targets of PPR56 identified in E. coli characterized by different RNA coverages and editing efficiencies (A) were selected for cloning in tandem behind the native nad4eU272SL target of PPR56 (B) or separately into the upstream MCS in the 5’-UTR (C). Editing efficiencies for off-targets in E. coli transcripts are given in brackets. Enhancement of RNA editing was found for three of the off-targets (cydCeU980PL, rarAeU407TI and fdhEeU403Q*) in either cloning arrangement and also for the, hitherto hypothetical, candidate editing cox3eU290SF when cloned downstream of nad4eU272SL (B).